US11040999B2 - Angiogenesis-inhibiting peptide and composition for preventing and treating angiogenesis-related disease comprising same as active ingredient - Google Patents

Angiogenesis-inhibiting peptide and composition for preventing and treating angiogenesis-related disease comprising same as active ingredient Download PDF

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US11040999B2
US11040999B2 US15/777,541 US201615777541A US11040999B2 US 11040999 B2 US11040999 B2 US 11040999B2 US 201615777541 A US201615777541 A US 201615777541A US 11040999 B2 US11040999 B2 US 11040999B2
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peptide
rlye
angiogenesis
arg
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US20190315803A1 (en
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Young Myeong KIM
Yi Yong BAEK
Won Jin PARK
Jeong Hun Kim
Dong Hyun JO
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Avixgen Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to improved peptides for inhibiting angiogenesis, such as Ac-RLYE (SEQ ID NO: 1) and R(D)LYE (SEQ ID NO: 6), and a composition for preventing and treating an angiogenesis-related disease, which contains the peptide as an active ingredient, and more particularly, to a peptide for inhibiting angiogenesis in which N-terminal Arg is acetylated in a peptide consisting of the sequence of amino acids (Arg-Leu-Tyr-Glu (SEQ ID NO: 1)), a peptide for inhibiting angiogenesis in which L-Arg is substituted with D-Arg in a peptide consisting of the sequence of amino acids (Arg-Leu-Tyr-Glu (SEQ ID NO: 6)), and a technique of preventing and treating a disease (cancer, diabetic retinopathy or senile macular degeneration) caused by excessive angiogenesis using a composition containing these peptides as an active ingredient.
  • Angiogenesis occurs under both normal and pathological conditions and refers to generation of a new blood vessel from an existing blood vessel. All types of angiogenesis occurring under physiological conditions such as embryo development and wound healing and pathological conditions such as cancer growth and the occurrence of retinal diseases are regulated by a balance between angiogenesis-promoting and inhibiting factors. However, abnormal angiogenesis caused by excessive generation and accumulation of angiogenesis-promoting factors under physiological conditions is the cause of several diseases including tumor growth and metastasis, rheumatoid arthritis, diabetic retinopathy and senile macular degeneration.
  • Angiogenesis is commonly induced by a sequential process of activation, proliferation, migration and tube formation of vascular endothelial cells, which is caused by various angiogenesis-promoting factors.
  • a vascular endothelial growth factor (VEGF) of the angiogenesis-promoting factors serves to induce proliferation, migration and differentiation of endothelial cells by activating various signaling cascades.
  • VEGF induces abnormal angiogenesis to promote the growth of tumor cells and retinal cells and vascular leakage, resulting in the growth and metastasis of tumors, diabetic retinopathy, and senile macular degeneration.
  • onset of the formation of new cancer vessels and retina vessels may be controlled by interfering with the biological activity and signaling transduction of VEGF using a neutralizing antibody and a signaling inhibitor of VEGF.
  • Angiogenesis inhibiting drugs targeting VEGF or a VEGF receptor are excellent therapeutic strategies for effectively controlling pathological (abnormal) angiogenesis-related human diseases.
  • angiogenesis inhibiting antibodies, proteins and chemicals have been developed for treating excessive angiogenesis-related diseases including tumors, diabetic retinopathy and senile macular degeneration, and have been clinically used.
  • side effects such as hypertension and hemorrhage may occur, or therapeutic limitations such as low specificity and bioavailability, antigenicity and inadequate pharmacokinetics have been reported.
  • a small peptide is easily mass-produced, has no antigenicity, and high solubility and bioavailability, and thus is proposed as a good material for drug development.
  • the inventors have conducted various studies for developing peptides that can effectively inhibit angiogenesis induced by VEGF, resulting in the development of an Arg-Leu-Tyr-Glu (RLYE (SEQ ID NO: 2)) peptide effectively blocking the binding between VEGF receptor-2 and VEGF.
  • RLYE Arg-Leu-Tyr-Glu
  • This peptide exhibited an activity of effectively inhibiting angiogenesis induced by VEGF, and such activity was confirmed to be very useful as a therapeutic agent for a disease caused by excessive angiogenesis, particularly, cancer.
  • the RLYE peptide (SEQ ID NO: 2) is rapidly degraded in blood and do not have a long half-life, which need to be improved. If the half-life can be extended, a sustained effect can be expected, and thus the RLYE peptide (SEQ ID NO: 2) was determined to be more effective in treating a disease caused by excessive angiogenesis (cancer, diabetic retinopathy, or senile macular degeneration). Accordingly, a method for improving the half-life of the RLYE peptide (SEQ ID NO: 2) was studied.
  • the present invention provides a peptide for inhibiting angiogenesis (Ac-RLYE (SEQ ID NO: 1)) in which N-terminal L-Arg is acetylated in a peptide consisting of a sequence of amino acids Arg-Leu-Tyr-Glu (R-L-Y-E or RLYE (SEQ ID NO: 1)) and a peptide for inhibiting angiogenesis (R(D)LYE (SEQ ID NO: 6)) in which N-terminal L-Arg is substituted with D-Arg in a peptide consisting of a sequence of amino acids Arg-Leu-Tyr-Glu (SEQ ID NO: 6).
  • the two peptides may inhibit angiogenesis induced by VEGF.
  • the present invention provides a composition containing at least one of the peptides as an active ingredient to prevent and treat an excessive angiogenesis-related disease.
  • the present invention provides a method for treating an angiogenesis-related disease, the method including administering at least one of the peptides to a subject.
  • the present invention provides a method for preventing an angiogenesis-related disease, the method including administering at least one of the peptides to a subject.
  • the present invention provides a use of a composition containing at least one of the peptides as an active ingredient to prevent and treat an angiogenesis-related disease.
  • composition for preventing and treating an angiogenesis-related disease of the present invention may be used in preventing and treating one or more diseases selected from the group consisting of tumor growth and metastasis, diabetic retinopathy, senile macular degeneration, rheumatoid arthritis and psoriasis.
  • the present invention provides a composition containing at least one of the peptides as an active ingredient to prevent and treat cancer.
  • the present invention provides a method for treating cancer, the method including administering at least one of the peptides to a subject.
  • the present invention provides a method for preventing cancer, the method including administering at least one of the peptides to a subject.
  • the present invention provides a use of a composition containing at least one of the peptides as an active ingredient to prevent and treat cancer.
  • composition for preventing and treating cancer containing the peptide of the present invention may effectively prevent and treat various types of cancer, particularly, solid tumors.
  • the present invention provides a composition containing at least one of the peptides as an active ingredient to prevent and treat diabetic retinopathy.
  • the present invention provides a method for treating diabetic retinopathy, the method including administering at least one of the peptides to a subject.
  • the present invention provides a method for preventing diabetic retinopathy, the method including administering at least one of the peptides to a subject.
  • the present invention provides a use of a composition containing at least one of the peptides as an active ingredient to prevent and treat diabetic retinopathy.
  • the present invention provides a composition containing at least one of the peptides as an active ingredient to prevent and treat senile macular degeneration.
  • the present invention provides a method for treating senile macular degeneration, the method including administering at least one of the peptides to a subject.
  • the present invention provides a method for preventing senile macular degeneration, the method including administering at least one of the peptides to a subject.
  • the present invention provides a use of a composition containing at least one of the peptides as an active ingredient to prevent and treat senile macular degeneration.
  • the peptide of the present invention may be used in drugs, foods or feed for humans or animals.
  • the peptide according to the present invention may be formulated according to a standard for formulating a conventional pharmaceutical preparation and a standard for formulating health supplement food of the Korean Food and Drug Administration (KFDA).
  • KFDA Korean Food and Drug Administration
  • the peptide of the present invention may be used as is, or in the form of a salt such as a pharmaceutically acceptable acid-addition salt or a metal complex, for example, a sodium (Na), potassium (K), calcium (Ca), zinc (Zn), or iron (Fe) salt.
  • a salt such as a pharmaceutically acceptable acid-addition salt or a metal complex, for example, a sodium (Na), potassium (K), calcium (Ca), zinc (Zn), or iron (Fe) salt.
  • the acid-addition salt may be hydrogen chloride, hydrogen bromide, a sulfate, a phosphate, a maleate, an acetate, a citrate, a benzoate, a succinate, a malate, an ascorbate, or a tartrate.
  • the peptide of the present invention may be diluted by mixing the oligopeptide with a pharmaceutically acceptable carrier by a conventional method according to an administration method, an administration type and a therapeutic purpose, or encapsulated in a container-like carrier.
  • the carrier When the carrier is used as a diluent, the carrier may be prepared in a formulation such as a powder, granules, injections, a syrup, a solution, tablets, suppositories, pessaries, ointment, cream or aerosol for oral administration or parenteral administration using at least one selected from the group consisting of a saline, a buffer, dextrose, water, glycerol, Ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose and ethanol.
  • the carrier is not limited to the above-mentioned types of carriers.
  • the parenteral administration refers to intravitreal, rectal, intravenous, intraperitoneal, intramuscular, intraaortic, transdermal or intranasal administration of an effective ingredient, or inhalation, other than oral administration.
  • the above-mentioned formulation may be prepared by further adding a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier or a preservative to induce rapid, sustained or extended release of an active ingredient after administration to a mammal.
  • a dosage may be adjusted according to a patient's condition, administration route and administration type, and it will be obvious to those of ordinary skill in the art that the dosage may vary in a wide range without limitation. Conventionally, it is determined that the peptide of the present invention can be continuously or intermittently administered daily at an experimentally effective amount of approximately 0.5 to 1.0 mg per kg of a body weight.
  • a peptide of the present invention has a long half-life, and a very excellent VEGF-induced angiogenesis inhibitory effect. Therefore, when the peptide developed in the present invention is used for excessive angiogenesis-related diseases, cancer, diabetic retinopathy or senile macular degeneration, these diseases can be very effectively prevented or treated.
  • FIG. 1 shows experimental results showing the stability of an RLYE peptide (SEQ ID NO: 2) in human serum.
  • NC negative control
  • CTRL physiological saline control (control).
  • FIG. 2 shows experimental results showing effects of RLYE (SEQ ID NO: 2) and modified RLYEs (“RLYE” disclosed as SEQ ID NO: 2) on stability in human serum and migration of vascular endothelial cells.
  • A An experimental result showing the stability of RLYE (SEQ ID NO: 2) and modified RLYEs (“RLYE” disclosed as SEQ ID NO: 2) (Ac-RLYE (SEQ ID NO: 1), RLYE-NH 2 (SEQ ID NO: 3), Ac-RLYE-NH 2 (SEQ ID NO: 4), and R(D)LYE (SEQ ID NO: 6)) in human serum
  • B An experimental result showing effects of RLYE (SEQ ID NO: 2) and modified RLYEs (“RLYE” disclosed as SEQ ID NO: 2) on half-life (t1 ⁇ 2) in human serum and inhibition (IC50) to migration of vascular endothelial cells.
  • B Discloses SEQ ID NOS 2, 1, 3-4 and 6, respectively, in order of appearance.
  • FIG. 3 shows experimental results showing effects of RLYE (SEQ ID NO: 2), Ac-RLYE (SEQ ID NO: 1) and R(D)LYE (SEQ ID NO: 6) on tube formation of vascular endothelial cells depending on pretreatment of human serum.
  • A An experimental result showing an effect of a peptide pretreated with phosphate-buffered saline (PBS) for three hours on tube formation of vascular endothelial cells induced by VEGF(V)
  • B An experimental result showing an effect of a peptide pretreated with human serum for three hours on tube formation of vascular endothelial cells induced by VEGF(V).
  • FIG. 4 shows effects of RLYE (SEQ ID NO: 2), Ac-RLYE (SEQ ID NO: 1) and R(D)LYE (SEQ ID NO: 6) on inhibition of tumor growth in human colorectal cancer cell-implanted mouse models.
  • C An experimental result showing an effect of administration of a peptide or bevacizumab (BVZ) on inhibition of tumor angiogenesis.
  • FIG. 5 shows experimental results showing effects of RLYE (SEQ ID NO: 2) and Ac-RLYE (SEQ ID NO: 1) on inhibition of choroidal neovascularization in laser-induced choroidal neovascularization mouse models.
  • B A quantitative analysis result for the area of choroidal neovascularization
  • C An experimental result showing an effect of a peptide on inhibition of choroidal neovascularization in retinal pigment epithelium/choroid/sclera complex tissue of an eyeball extracted after intravenous injection of fluorescein isothiocyanate (FITC)-binding dextran.
  • FITC fluorescein isothiocyanate
  • FIG. 6 shows experimental results showing effects of RLYE (SEQ ID NO: 2) and Ac-RLYE (SEQ ID NO: 1) on inhibition of retinal vascular leakage in diabetic mouse models.
  • A An experimental result showing an effect of a peptide on inhibition of retinal vascular leakage due to diabetes induced by streptozotocin (STZ).
  • B An experimental result showing an effect of a peptide on inhibition of retinal vascular leakage due to diabetes.
  • Serum was separated by centrifugation (tabletop centrifuge, 3000 rpm, 20 min) of blood obtained from a healthy person.
  • the serum was filtrated using a filter (Millipore) with a pore size of 0.22 ⁇ M, and then 50 ⁇ l of the human serum was mixed with 100 ⁇ g of RLYE (SEQ ID NO: 2), R(D)LYE (SEQ ID NO: 6), RLYE-NH 2 (SEQ ID NO: 3) or Ac-RLYE (SEQ ID NO: 1) (100 ⁇ g/50 ⁇ l, PBS, pH 7.4), and incubated by time at a constant temperature of 37° C.
  • RLYE SEQ ID NO: 2
  • R(D)LYE SEQ ID NO: 6
  • RLYE-NH 2 SEQ ID NO: 3
  • Ac-RLYE SEQ ID NO: 1
  • a peptide concentration of the sample was calculated by a peak area after the sample was separated by C18 reverse phase high-performance liquid chromatography (HPLC; Vydac protein and peptide C18 column, 0.1% trifluoroacetate in H 2 O for equilibration, and 0.1% trifluoroacetate in acetonitrile for elution).
  • HUVECs human umbilical cord vascular endothelial cells
  • a Transwell culture plate to which a polycarbonate filter (pore size: 8 ⁇ m) with a diameter of 6.5 mm was attached. A surface of the filter was coated with 10 ⁇ g of gelatin.
  • Fresh M199 medium 1% fetal bovine serum
  • VEGF 10 ng/ml
  • HUVECs (1 ⁇ 10 6 cells/100 ⁇ l) which had reacted with 0.15 nM or 1.5 nM peptides at room temperature for 30 minutes were carefully transferred to an upper compartment of the plate.
  • the resulting HUVECs were cultured in a CO 2 -constant temperature and humidity chamber for 4 hours, and the cells migrated under the Transwell filter were stained by H&E staining and quantified using an optical microscope.
  • a degree of tube formation of HUVECs was determined using growth factor-reduced Matrigel. 250 ⁇ l of the Matrigel (10 mg protein/ml) was added into a 24-well plate to allow polymerization for 30 minutes at 37° C. HUVECs cultured in M199 (1% fetal bovine serum) medium for 6 hours were carefully transferred onto the Matrigel in the culture plate at a density of 2 ⁇ 10 5 cells/well, and VEGF (10 ng/ml) were cultured alone or with peptides (0.15 nM) at 37° C. for 20 hours. Meanwhile, peptides that were reacted in PBS or fresh human serum for 3 hours were also used. The degree of tube formation of HUVECs was visualized using an inverted phase-contrast microscope, and a length of the formed tube was measured and quantified by Image-Pro Plus version 4.5 (Media Cybernetics, San Diego, Calif.).
  • HCT116 Human colon cancer cells (HCT116, 1 ⁇ 10 7 cells/100 ⁇ l) were subcutaneously injected into the left side of a nude mouse (BALB/c nu/nu, 6 week old, male), and then when a tumor volume reached at least 50 to 70 mm 3 (approx. 7 days), physiological saline (negative control), RLYE (SEQ ID NO: 2) (1.0 mg/kg), R(D)LYE (SEQ ID NO: 6) (1.0 mg/kg) or Ac-RLYE (SEQ ID NO: 1) (1.0 mg/kg; abnormal, experimental group) was intraperitoneally injected once a day.
  • a VEGF neutralizing antibody, bevacizumab was intraperitoneally injected at 2 mg/kg twice a week (positive control).
  • a tumor size was two-dimensionally measured using calipers.
  • a tumor volume (mm 3 ) was calculated by the formula: width 2 ⁇ length ⁇ 0.52.
  • Tumor tissue was fixed with 10% formalin, and inserted into paraffin.
  • a slide was prepared using a tumor tissue section, immersed in xylene to remove paraffin, and rehydrated by stepwise treatment with 100%, 95%, 80%, and 70% ethanol. Afterward, the slide was washed with deionized water several times at room temperature.
  • the tissue section was reacted in 0.3% hydrogen peroxide-containing methanol for 15 minutes, and washed with PBS three times.
  • the tissue section was reacted in 3% goat serum-containing PBS for 2 hours at room temperature, and reacted with FITC-isolectin B4 (5 mg/ml; Vector Laboratories) for 1 hour.
  • the tissue section was washed with PBS three times and then a permanent slide was prepared using a cryo-mount solution (OCT mouting medium, Tissue Tek, Sakura Finetek), and then tumor vessels were visualized using a fluorescence microscope.
  • a Bruch membrane between a retinal layer and a choroidal layer was destroyed by irradiating the mouse retina with a diode laser at an intensity of 400 mW and a duration of 50 ms, and then choroidal neovascularization occurred over 14 days.
  • the retinas of 6 week-old male C57BL/6 mice were irradiated with a laser under the above conditions, the formation of a lesion was confirmed through a safety inspection on day 10, and 1 ⁇ l of a 1.5 mM EYLR (SEQ ID NO: 5), RLYE (SEQ ID NO: 2) or Ac-RLYE (SEQ ID NO: 1) solution was intravitreally injected.
  • the average of the choroidal neovascular areas was represented as 100%, and a therapeutic effect of each treatment group was analyzed.
  • 1 ml (1.25 mg) of FITC-dextran (250 kDa) was injected into the left ventricle one hour before extraction of the mouse eyeball to allow systemic perfusion. After extraction, the eyeball was fixed in 4% paraformaldehyde for 1 hour. Then, the cornea and the crystalline lens were cut off by scissors, the retina was carefully removed using forceps to prepare a complex of retina pigment epithelium/choroid/sclera tissues. The retina flat-mounted complex tissue was observed under a fluorescence microscope and photographed.
  • a freshly prepared STZ solution (100 mM) in a citrate buffer (100 mM, pH 4.5) was intraperitoneally injected at 150 mg/kg into the mice, and to prevent hypoglycemic shock, 10% sucrose was sufficiently provided.
  • blood glucose was measured using an Accu-Chek Performa blood glucose meter (Roche Diagnostics GmbH, Germany), and when non-fasting blood glucose within 1 to 2 weeks was maintained at 300 mg/dl or more, the mice were used as diabetic animal models.
  • mice The diabetes-induced mice were anesthetized with 2% avertin, and 1 ⁇ l of a 1.5 mM EYLR (SEQ ID NO: 5), RLYE (SEQ ID NO: 2) or Ac-RLYE (SEQ ID NO: 1) solution was intravitreally injected.
  • EYLR EYLR
  • RLYE SEQ ID NO: 2
  • Ac-RLYE SEQ ID NO: 1
  • the peptide was injected, and after 24 hours, 1 ml (1.25 mg) of FITC-dextran (250 kDa) was injected into the left ventricle of each mouse to allow circulation for approximately 5 minutes.
  • the mice were euthanized by cervical dislocation, and the eyeballs were extracted and fixed in 4% paraformaldehyde at room temperature for 1 hour.
  • the retina was isolated from the fixed eyeball to prepare a retinal flat mount, and vascular leakage was observed under a confocal microscope. Meanwhile, the fluorescence of FITC-
  • RLYE (SEQ ID NO: 2) was added to each of PBS and human serum, and incubated by time at a constant temperature of 37° C.
  • RLYE (SEQ ID NO: 2) was incubated in PBS at a constant temperature, degradation of the RLYE peptide (SEQ ID NO: 2) did not occur, and when RLYE peptide (SEQ ID NO: 2) was incubated in human serum, the half-life of RLYE (SEQ ID NO: 2) was 1.2 hours (refer to FIG. 1 (A)).
  • RLYE a major component of serum, albumin, and inhibitors of various proteases were added to RLYE (SEQ ID NO: 2), followed by incubation at a constant temperature of 37° C.
  • the albumin did not induce RLYE (SEQ ID NO: 2) degradation, and it was confirmed that RLYE (SEQ ID NO: 2) degradation due to human serum was not inhibited by treatment of aprotinin, EDTA, leupeptin and phenylmethyl sulfonyl fluoride (PMSF), which are known as protease inhibitors (refer to FIG. 1 (B)).
  • PMSF phenylmethyl sulfonyl fluoride
  • RLYE SEQ ID NO: 2
  • modified RLYE SEQ ID NO: 2
  • Ac-RLYE SEQ ID NO: 1
  • RLYE-NH 2 SEQ ID NO: 3
  • Ac-RLYE-NH 2 SEQ ID NO: 4
  • R(D)LYE SEQ ID NO: 6
  • IC50 values of RLYE (SEQ ID NO: 2), Ac-RLYE (SEQ ID NO: 1), RLYE-NH 2 (SEQ ID NO: 3), Ac-RLYE-NH 2 (SEQ ID NO: 4) and R(D)LYE (SEQ ID NO: 6) were 0.08, 0.05, 0.11, 80.2 and 0.06 nM, respectively, and it was confirmed that the angiogenesis inhibitory effects of Ac-RLYE (SEQ ID NO: 1) and R(D)LYE (SEQ ID NO: 6) were similar to or a little better than that of RLYE (SEQ ID NO: 2) which was confirmed to have an angiogenesis inhibitory effect at an early stage (refer to FIG. 2 (B)).
  • RLYE SEQ ID NO: 2
  • Ac-RLYE SEQ ID NO: 1
  • R(D)LYE SEQ ID NO: 6
  • each peptide was injected into the peritoneal cavity once a day at a dose of 1.0 mg/kg/day, and 2.0 mg/kg of a clinically-used VEGF neutralizing antibody anticancer agent, bevacizumab (Avastin), as a positive control, was intraperitoneally injected twice a week.
  • a clinically-used VEGF neutralizing antibody anticancer agent bevacizumab (Avastin)
  • a laser-induced choroidal neovascularization inhibitory effect is generally studied in mouse models.
  • EYLR reverse-sequence of the RLYE peptide (SEQ ID NO: 2)
  • RLYE SEQ ID NO: 2
  • Ac-RLYE SEQ ID NO: 1

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US15/777,541 2015-12-10 2016-07-05 Angiogenesis-inhibiting peptide and composition for preventing and treating angiogenesis-related disease comprising same as active ingredient Active US11040999B2 (en)

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